Reznikoff C A, DeMars R
Cancer Res. 1981 Mar;41(3):1114-26.
We have established and characterized a diploid cell strain of normal human endothelial cells, RuBa 7E. RuBa 7E cells have an average cloning efficiency of 20% during early passages and undergo approximately 50 doublings in vitro before senescing spontaneously. At confluence, RuBa 7E cells form a homogeneous monolayer of flat polygonal cells. RuBa 7E cells react positively with antibody to human endothelial-specific Factor VIII. The toxic and mutagenic effects of N-methyl-N'-nitro-N-nitrosoguanidine on RuBa 7E cells were studied and are similar to those reported for diploid human fibroblasts. Mutant cells lacking hypoxanthine-guanine phosphoribosyltransferase were selected by their resistance to 6-thioguanine. The spontaneous incidence of mutants was less than or equal to 6 X 10(-6), and the induced incidence was 4.4 X 10(-4) at a survival frequency of 0.05. All 17 mutants that were tested lacked detectable hypoxanthine-guanine phosphoribosyltransferase activity, and none grew in medium containing azaserine and hypoxanthine. Autoradiography showed that mutant cells incorporated radioactive adenine but did not incorporate radioactive hypoxanthine. Unlike human fibroblasts, in which the recovery of 6-thioguanine-resistant mutants is reduced by contact feeding when the inoculum size during selection is increased above 10(4) cells per P60 dish, 5 to 10 X 10(4) RuBa 7E cells can be plated per P60 dish without reducing mutant recovery. This apparent lack of plating density suppression of mutant recovery makes RuBa 7E cells a comparatively compact and economical system for quantifying mutagenesis in diploid human cells. In order to determine whether RuBa 7E cells would undergo a distinct morphological transformation toward cancer in vitro, we infected them with SV40. As early as 14 days postinfection, discrete foci of morphologically transformed, mitotically active cells were seen against a monolayer background of normal cells when cultures were maintained in medium with low serum. Seven of the 33 foci which were obtained were studied for SV40-specific viral T-antigen, and all were positive. The facility with which RuBa 7E cells can be mutagenized and the ease with which morphological transformants can be identified make these cells potentially useful for studies comparing the mutagenic and transforming effects of chemicals and other agents on diploid human cells.
我们已经建立并鉴定了一株正常人内皮细胞的二倍体细胞株RuBa 7E。RuBa 7E细胞在早期传代过程中的平均克隆效率为20%，在体外自发衰老前可经历约50次倍增。汇合时，RuBa 7E细胞形成扁平多边形细胞的均匀单层。RuBa 7E细胞与人内皮特异性因子VIII抗体呈阳性反应。研究了N-甲基-N'-硝基-N-亚硝基胍对RuBa 7E细胞的毒性和诱变作用，其结果与二倍体人成纤维细胞的报道相似。通过对6-硫鸟嘌呤的抗性筛选出缺乏次黄嘌呤-鸟嘌呤磷酸核糖转移酶的突变细胞。突变体的自发发生率小于或等于6×10^(-6)，在存活频率为0.05时，诱导发生率为4.4×10^(-4)。检测的17个突变体均未检测到次黄嘌呤-鸟嘌呤磷酸核糖转移酶活性，且在含有重氮丝氨酸和次黄嘌呤的培养基中均不能生长。放射自显影显示突变细胞摄取放射性腺嘌呤，但不摄取放射性次黄嘌呤。与人类成纤维细胞不同，当选择过程中的接种量增加到每P60培养皿超过10^4个细胞时，接触饲养会降低6-硫鸟嘌呤抗性突变体的回收率，而每P60培养皿接种5至10×10^4个RuBa 7E细胞不会降低突变体回收率。这种明显不存在的接种密度对突变体回收率的抑制作用，使得RuBa 7E细胞成为一种相对紧凑且经济的系统，用于定量二倍体人类细胞中的诱变作用。为了确定RuBa 7E细胞在体外是否会向癌细胞发生明显的形态转化，我们用SV40感染了它们。早在感染后14天，当培养物在低血清培养基中维持时，在正常细胞的单层背景上可以看到形态转化、有丝分裂活跃的细胞形成的离散集落。对获得的33个集落中的7个进行了SV40特异性病毒T抗原的研究，结果均为阳性。RuBa 7E细胞易于诱变以及易于鉴定形态转化体的特性，使得这些细胞对于比较化学物质和其他试剂对二倍体人类细胞的诱变和转化作用的研究具有潜在的用途。
